Process for producing protein microparticles

ABSTRACT

The present invention relates to a process for producing protein microparticles in dilute organic acid solutions and in the absence of an alcohol such as ethanol. The microparticles are formed by dissolving a cereal prolamin protein in a concentrated organic acid solution with agitation and then diluting the solution with an aqueous solution. Protein microparticles having vacuoles are thus formed. The protein microparticles may be used to form powders, films, coatings, matrices, scaffolds and the like. Complete films can be formed from the protein microparticles of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national phase entry of international patentapplication No. PCT/IB2009/054386 filed Oct. 7, 2009, which claimspriority to ZA application No. 2008/08528 filed Oct. 7, 2008. Thedisclosure of said applications is hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

The invention provides a process for producing protein microparticlesthat can be used for micro-encapsulation, matrix or scaffold formationor formed into films or coatings.

A number of processes are known for making microspheres ormicroparticles from proteins for a variety of applications, such as foodcoatings, drug delivery and delayed release of pesticides, fertilizersand agents for environmental cleanup. However, non acidic organicsolvents such as ethanol or acetone are often used to dissolve theprotein. These solvents are often incompatible with and difficult toremove from food, and there has therefore been a reluctance by the foodand pharmaceutical industries to use protein film or microparticlesystems. Moreover, the use of non acidic organic solvents poses safetyissues with the emission of vapours, the fire hazard that they pose, andthe possible residuals that they may leave in the food orpharmaceuticals. Many processes also require the use of elevatedtemperatures.

U.S. Pat. No. 5,736,178 teaches that film forming colloidal dispersionscan be made from dilute aqueous acid solutions of gluten-derived proteinby causing the protein to precipitate as microparticles. However, theprotein has first to be dissolved in an alcohol such as ethanol, whichleads to the disadvantages listed above.

The applicant has therefore identified a need for a process forproducing protein microparticles in the absence of non acidic organicsolvents such as ethanol.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided aprocess for making protein microparticles, the process including thesteps of:

(a) dissolving cereal prolamin protein in an organic acid with agitationand in the absence of an alcohol; and

(b) diluting the solution containing the protein with an aqueoussolution and thereby forming protein microparticles having vacuoles.

The protein may be any cereal prolamin protein, such as wheat gluten,barley hordein, maize zein or sorghum kafirin.

The organic acid may be propionic, lactic or acetic acid, or any othersuitable acid.

An additive, such as a plasticizer, colouring agent, flavouring agent,preservative, trace mineral, nutrient, enzyme, hormone, nutraceuticalprobiotic, prebiotic, drug or any combination thereof, may be added toeither of steps (a) or (b).

In step (a) the solution of the protein is preferably saturated orhighly concentrated.

In step (a), the dissolution is carried out with agitation so as toensure that the protein microparticles formed have vacuoles. Theagitation may be provided by stirring, shaking, homogenisation, bubblinggas through the solution or the like.

In step (b), the solution containing the protein preferably is dilutedwith water to a final concentration of 2 parts by weight of the proteinto 2 to 30 parts by weight of the organic acid to 96 to 68 parts byweight of water, thereby forming the protein microparticles.

The process may be carried out at a temperature of from about 20° C. to40° C., and more particularly, at room temperature.

The protein may be modified, either physically, chemically orenzymically, prior to or after dissolution, to enhance or alter thefunctional properties of the microparticles or products such as filmsformed therefrom. For example, the protein may be crosslinked withtannins or enzymes and/or heated.

The protein microparticles so produced may be used to make films,coatings or matrices as described below.

According to a second aspect of the invention, there is provided aprocess for making a complete film or coating from the proteinmicroparticles produced as described above, comprising the steps of:

(i) preparing a suspension of the protein microparticles in an aqueoussolution of an organic acid to a final concentration of 2 parts byweight of the protein to 2 to 60 parts by weight of an organic acid to96 to 38 parts by weight of water; and

(ii) drying that suspension to form the film or coating.

The film or coating may be clear or cloudy and transparent ortranslucent.

According to a third aspect of the invention, there is provided aprocess for making a matrix from the protein microparticles, comprisingthe steps of:

-   -   (i) preparing a suspension of the protein microparticles in an        aqueous solution of an organic acid to a final concentration of        2 parts by weight of the protein to 2 to 60 parts by weight of        an organic acid to 96 to 38 parts by weight of water;    -   (ii) washing away the organic acid; and    -   (iii) drying the suspension to form the matrix.

The processes which form the second and third aspects of the inventionare preferably carried out in the absence of any non acidic organicsolvents, in particular alcohols such as ethanol.

The organic acid may be propionic or acetic acid for the making of filmsor coatings, or propionic, lactic or acetic acid for the making ofmatrices, or any other suitable acid.

The final concentration of the suspension may be 2 parts by weight ofthe protein to 10 to 60 parts by weight of the organic acid to 88 to 38parts by weight of water.

When the organic acid used is acetic acid, the final concentration ispreferably 2 parts by weight of the protein to 20 to 60 parts by weightof acetic acid to 78 to 38 parts by weight of water, more preferably 2parts by weight of the protein to 20 to 30 parts by weight of aceticacid to 78 to 68 parts by weight of water, most preferably 2 parts byweight of the protein to 21.6 parts by weight of acetic acid to 76.4parts by weight of water.

However, when the organic acid is acetic acid and the protein is insolution having been extracted from brewers spent grain or otherco-products of alcohol production from cereals, then the ratio ofprotein to acid to water is preferably 2 parts protein to 2 parts aceticacid to 96 parts of water.

When the organic acid used is propionic acid or lactic acid, the finalconcentration is preferably 2 parts by weight of the protein to 10 to 30parts by weight of propionic acid or lactic acid to 88 to 68 parts byweight of water, more preferably 2 parts by weight of the protein to 10to 15 parts by weight of propionic acid or lactic acid to 88 to 83 partsby weight of water, most preferably 2 parts by weight of protein to 10.8parts by weight of propionic acid or lactic acid to 87.2 parts by weightof water.

In the processes of the second and third aspects of the invention, thesuspension prepared in step (i) is preferably allowed to stand for 8 to24 hours prior to step (ii).

Further, before step (ii), there is preferably added to the suspension aplasticizer in an amount of 0.2 to 0.6 parts by weight of theplasticizer to 1 part by weight of the protein, more preferably about0.4 parts by weight of the plasticizer to 1 part by weight of theprotein.

Examples of plasticizers include glycerol, polyethylene glycol,polypropylene glycol and lactic acid used alone or in combination,dibutyl tartrate, monoglycerides such as oleic acid, palmitic acid orstearic acid, triethylene glycerol and sorbitol, or any other suitableplasticizer or combination thereof.

A preferred plasticizer is a mixture of 1:1:1 (w/w) glycerol:polyethylene glycol (400): lactic acid.

Other additives such as colouring agents, flavouring agents,preservatives, trace minerals, nutrients, enzymes, hormones,nutraceuticals, pharmaceuticals, probiotics, prebiotics, drugs andcombinations may be added before step (ii) or (iii).

For the preparation of a film, the suspension of step (i) may be castdirectly onto a casting surface. Thereafter the liquid phase is removedby drying, either with or without the application of heat, to give acomplete film. The film may be clear or cloudy, transparent ortranslucent.

For the preparation of a coating on a substrate, the suspension of step(i) may be used to cover a surface of the substrate. Thereafter theliquid phase is removed by drying, either with or without theapplication of heat, to give a complete coating on the substrate. Thecoating may be clear or cloudy, transparent or translucent.

For the preparation of a matrix incorporating a compound or substance,the suspension of step (i) has added to it the compound or substance.Thereafter the liquid phase is removed by drying, either with or withoutthe application of heat, to give an opaque matrix encapsulating orincorporating the compound or substance.

According to a fourth aspect of the invention, the proteinmicroparticles may be formed according to the process of the firstaspect of the invention so as to incorporate or encapsulate a compound,such as a nutrient, vitamin, enzyme, hormone, pharmaceutical,plasticizer, preservative, colouring agent, flavouring agent, tracemineral, nutraceutical, probiotic or prebiotic, or combinations thereof.The compound may be added to either of the concentrated acid solution orthe aqueous solution prior to the formation of the microparticles.

Alternatively, the solution or dispersion containing the proteinmicroparticles may be dried to form a powder.

In this case, according to a fifth aspect of the invention, there isprovided a process of making a coating on a substrate from the driedprotein microparticles, including the steps of:

-   -   (i) mixing the dried protein microparticles with a dry substrate        to be coated;    -   (ii) adding an amount of a concentrated organic acid to the        mixture of step (i) so that the protein microparticles coat the        substrate; and    -   (iii) removing the organic acid to fuse the protein        microparticles to the substrate to form the coating.

The organic acid may be removed by evaporation, optionally with the aidof heat.

According to a sixth aspect of the invention, there is provided aprocess of incorporating a compound into a matrix formed from theprotein microparticles including the steps of:

-   -   (i) mixing the dried protein microparticles with a solution of        the compound to be incorporated, the dried protein        microparticles being substantially insoluble in the solution;        and    -   (ii) removing the solvent, leaving the compound incorporated        into the matrix.

The solvent may be removed by evaporation, optionally with the aid ofheat.

According to a seventh aspect of the invention, there is provided aprotein microparticle formed from the first process described above. Themicroparticle must have vacuoles and thus a large internal surface area,and may be softer than a microparticle formed using a non acidic organicsolvent, such as ethanol.

According to an eighth aspect of the invention, there is provided anaqueous solution or dispersion of microparticles produced by the firstprocess described above.

According to a ninth aspect of the invention, there is provided a drypowder of protein microparticles produced by the first process describedabove.

According to a tenth aspect of the invention, there is provided a filmcast from a dispersion of microparticles produced by the second processdescribed above.

According to a eleventh aspect of the invention, there is provided asubstrate having a coating of microparticles produced by the second orfifth process described above.

The substrate may be selected from the group consisting of foodstuff,fruits, vegetables, minimally processed and processed fruits andvegetables, seeds, nuts, animal feed products, colouring agents,flavouring agents, trace minerals, nutrients, enzymes, hormones,pharmaceuticals nutraceuticals, probiotics, prebiotics, drugs and otherpharmaceutical products, medical devices and the like, and combinationsthereof.

The starting protein may be a commercially available protein such ascommercial zein.

Alternatively the protein can be provided by extracting protein fromwhole grain (sorghum, maize, wheat, barley, etc. or mixtures thereof) ora co-product such as flour, bran, spent grain, distiller's dry grainwith solubles or other co-products of alcohol production from cereals,with an organic acid such as glacial acetic acid, lactic acid orpropionic acid, and removing the residual solid material. Mixtures ofproteins can also be used for extraction such as those found in brewersspent grain, distiller's dry grain with solubles or other co-products ofalcohol production from cereals, which may be mixtures of, but not tothe exclusion of other cereal combinations, for example, sorghum andmaize in various proportions or barley and maize.

This protein solution can then be used directly in step (b) of theprocess of the first aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Scanning Electron Micrograph of kafirin microparticlesproduced by the prior art method of Parris, Cook and Hicks (2005) JAgric Food Chem 53: 4788-4792, using aqueous ethanol to dissolve thekafirin prior to microparticle formation.

FIG. 2 shows micrographs of kafirin microparticles produced according tothe process of the present invention, at different acid concentrations:a-c Light Micrographs at 5.4%, 21.6% and 40% acetic acid, respectively;d-f Scanning Electron Micrograph at 5.4%, 21.6% and 40% acetic acid,respectively; g-i Transmission Electron Micrographs at 5.4%, 21.6% and40% acetic acid, respectively; j-l Transmission Electron Micrographshigher magnification 5.4%, 21.6% and 40% acetic acid, respectively.

FIG. 3 shows a Scanning Electron Micrograph of freeze-dried kafirinmicroparticles produced according to the process of the presentinvention.

FIG. 4 shows: (a): a 2% kafirin microparticle film cast from 21.6%acetic acid, the film being approximately 15 microns thick, flexible,colourless, clear and transparent with a smooth surface; and (b): a 2%kafirin film cast from glacial acetic acid, the film being approximately30 microns thick, flexible, almost colourless, clear, not completelytransparent with a rough surface.

FIG. 5 shows the effect of kafirin microparticles made with differentorganic acids with increasing acid concentration on film formation, a-cacetic acid, 5.4%, 10.8%, 21.6%, d-f propionic acid, 5.4%, 10.8%, 21.6%,respectively. a and b show that complete, clear films cannot be formedat either 5.4% or 10.8% acetic acid, respectively. The films are opaque,fragmented and rough on the surface. In contrast, at 21.6% acetic acid acomplete, colourless, clear, transparent and smooth film is formed (c).d shows that with 5.4% propionic acid a complete film cannot be formed.The film is opaque and fragmented with a rough surface but less so thanin a and b where acetic acid is used at 5.4% and 10.8% respectively. eand f show that at 10.8% and 21.6% propionic acid, respectively, acomplete, colourless, clear, transparent and smooth film is formed.

FIG. 6 shows films and coatings made from microparticles made directlyfrom an extract of sorghum brewers spent grains. A-20% acetic acid,B-10% acetic acid, C-2% acetic acid, D-1% acetic acid; 1-2% kafirin,2-1% kafirin, 3-0.5% kafirin. Note all the films are complete.

FIG. 7 shows minimally processed avocados stored for 5 days at 4° C. A:uncoated avocados, B: avocados coated with a kafirin microparticlecoating formulation according to the invention.

DETAILED DESCRIPTION

The present invention relates, in a first aspect, to a process forproducing dispersions of protein microparticles in dilute organic acidsolutions. The microparticles are formed by dissolving the protein in aconcentrated organic acid solution and then diluting the solution withan aqueous phase.

The formation of the microparticles is dependant on the relativesolubility of the protein in the organic acid solution. As the organicacid concentration is reduced by the addition of an aqueous phase, theprotein is no longer soluble and therefore comes out of solution,forming microparticles. A compound may be added to the solution prior tothe formation of the microparticles so as to be incorporated into, orencapsulated by, the microparticles. The dispersion of microparticles indilute organic acid is stable and homogeneous under ambient storageconditions without microbial contamination. The microparticles can beseparated from the dilute organic acid, washed and dried to form apowder, or may be used to form a continuous film or to coat a substrate.

As used herein, a concentrated acid is generally regarded as having aconcentration of 80% acid or greater, and a dilute acid is generallyregarded as having a concentration of less than 80% acid.

No alcohol, such as ethanol, is used in the process and all theconstituents can be food compatible, rendering the microparticlessuitable for consumption by humans and animals. Unlike known processesfor forming microparticles, the present process does not requireelevated temperatures, and can be performed at temperatures ranging from20° C. to 40° C., such as at room temperature. Toxic chemicals are alsonot required.

Particle size and definition. As used herein, ‘micro’ refers to aparticle with a diameter ranging from nanometers to micrometers.Microspheres are usually considered to be spherical particles, whereasmicroparticles are usually slightly larger and irregular ornon-spherical in shape. In the present invention, the microparticles maybe either spherical or of irregular shape, but both will be referred toas microparticles. The term ‘microparticle’ is therefore intended to beinclusive of microspheres.

The size of the microparticles can range from nano sized (nanometers) tomicrometers but more particularly are between 1-10 microns for sphericalparticles and can be somewhat larger for irregular shaped particles ormicroparticle aggregates, which may be up to several millimeters. Thesize can, however, be manipulated to produce larger or smallerparticles, depending on the method of preparation, e.g. variation ofrate of dilution or shear applied during preparation.

The formation of microparticles with a range of sizes allows formultiple applications. This includes food uses, e.g. film formation forfood packaging, edible coatings for the prolongation of shelf-life offood products, and encapsulation of, for example, foodstuffs, animalfeed products, food ingredients, enzymes, hormones, colouring agents,flavouring agents, trace minerals, nutrients, pharmaceuticals,nutraceuticals, probiotics, and prebiotics. Other non-food uses couldinclude drug delivery, other medical uses such as tissue engineering,tissue matrixes or scaffolds and coatings for cardiovascular and otherbiomedical devices, biological semi-conductors and delayed release ofpesticides and fertilizers.

The microparticles made by the process of this invention have a roughporous surface and numerous internal holes or vacuoles as shown byScanning Electron Microscopy (outer surface) and Transmission ElectronMicroscopy (internal structure) (FIG. 2). This method of preparationthus results in the formation of microparticles with a very largesurface area.

Microparticles produced by this present invention have a rough pittedexternal surface, appear to be soft but may be hardened by cross-linkingand will form continuous films, whereas microparticles produced by theprior art using aqueous ethanol have an almost smooth external surface,appear to be firmer and do not form continuous films.

Proteins for forming microparticles. The proteins are cereal prolaminproteins, and more particularly (but not exclusively), kafirin, gluten,hordein or zein or mixtures thereof. Typically, the protein is kafirin.Proteins are used to make microparticles since they are natural, havediverse properties, which can be modified, and are degradable both invitro and in vivo to amino acids and small peptides. They are thereforesuitable for human and animal consumption and for pharmaceuticaladministration. Hydrophobic proteins have limited solubility in waterbut are soluble in organic solvents, aqueous mixtures of organicsolvents, binary solvents and solvents with extreme pHs, such as acidsor bases. Kafirin is the preferred prolamin protein since it is morehydrophobic than other prolamins such as wheat gliadin and maize zein,and is also more crosslinked than zein, resulting in better functionalproperties.

The protein may be a protein isolate or may be extracted from aco-product material, such as brewers spent grain, distiller's dry grainwith solubles or other co-products of alcohol production from cereals.

Acids used to make the microparticles. The protein is dissolved in anappropriate organic acid. The protein is usually considered soluble if0.5% (w/v) of the protein dissolves in a solvent to form a clearsolution at ambient temperature (20-25° C.) (Shukla, R. and Cheryan, M.(2001) Zein: The Industrial Protein From Corn, Industrial Crops andProducts 13:171-192). Examples of organic acids which can be used todissolve the proteins are acetic acid, lactic acid, and propionic acid.The use of these acids confers the advantage of food compatibility tothe microparticles. In addition, the microparticles formed in this wayare microbially stable and no additional preservatives are needed. Thepreferred acid in the process of this invention is acetic acid.

Powder formation. The microparticles can be separated from the diluteorganic acid solution in which they are formed and washed with anaqueous solution. The aqueous solution is then removed and themicroparticles dried to a powder. The method of separation can be, forexample, filtration or centrifugation. The method of drying can includelyophilization, flash drying, spray drying, fluid bed drying or anyother suitable drying method. The methods of separation and drying willtypically be dependant on the protein used. The powder can be stored andhandled without refrigeration or other special handling techniques.Re-hydration can be achieved by adding the powder to an dilute organicacid solution with agitation sufficient to re-suspend the particles. Theamount of dilute organic acid solution used for reconstitution isdependant on the concentration of the final product required and the usefor which it is required.

Microparticles which have been dried retain their structure. They arespherical or irregular in shape with a rough pitted surface, as shown byScanning Electron Microscopy (FIG. 3).

Film and coating formation. When the protein microparticles are to beused to form a free-standing film, the dilute organic acid suspension ofthe microparticles can be cast directly onto a casting surface. Theliquid phase is then removed by drying, either with or without theapplication of heat. A complete (as opposed to fragmented, cracked orwith holes) film is produced of variable thickness, depending on theconcentration of protein used to make the microparticles (FIG. 4 a). Thefilm will typically have a thickness of 10-30 microns. The film may beused as a packaging material, such as for foodstuffs, food ingredientsand pharmaceuticals for veterinary or human use. The film may also beused as an interleaving material, such as for separating portions of thesame food product, for example pizza, or between different components inthe same food product, such as the fruit filling and pastry in a pie toprevent moisture transfer. The film may be clear or cloudy.

When a coating is required on a substrate, the aqueous suspension of themicroparticles is used to cover a surface of the substrate and theaqueous phase is then allowed to evaporate, forming a coating on thesubstrate surface. The substrate may be that of an edible foodstuff orfood ingredient, which is sensitive to oxidation or moisture loss, suchas a fruit, nut, spice or vegetable, either whole or minimally processedor processed or an animal or fish feed product. Other suitable productscould also be coated, such as a flower, pharmaceutical compound,enzymes, hormones, tablet compositions, or other pharmaceutical,biological or medical products, nutrients, nutraceuticals, probiotics,prebiotics and the like, or combinations thereof.

It is important that the suspension of protein microparticles is dilutedas set out in relation to the second and third processes of theinvention, so as to produce films and coatings which are complete. Thisdistinguishes the films and coatings from those made by prior artprocesses, using ethanol or different proportions ofprotein:solvent:water, which may not be complete. The films preparedaccording to the invention may also be clear or cloudy, transparent ortranslucent.

The dispersion of protein microparticles can be co-formulated withvarious additives, such as plasticizers, which improve the functionalproperties of films and coatings made from the microparticle dispersion.Films produced from protein microparticles are complete and can be madethinner than other films, such as from 10 μm to 30 μm, and preferablyabout 15 μm. The films may also have some superior functional propertiesto films produced by ethanolic solutions of proteins, such as waterbarrier properties and being clear, transparent or translucent (othercast films are often fragmented and opaque).

Encapsulation within matrices. When the protein microparticles to beformed are to be used to encapsulate a compound, the compound can beincorporated into the aqueous solution, organic acid or proteinsolution, or a combination thereof, prior to the formation of themicroparticles. When the resulting solutions are mixed with agitation,microparticles are formed containing the compound which is to beencapsulated.

Another way to effect encapsulation of a compound into themicroparticles is by mixing the previously dried microparticles of theinvention with a substance in solution or suspension in which themicroparticles are not soluble. On evaporation of such solvent thecompound remains encapsulated within the microparticles.

Microparticle modification. The properties of the microparticles can bemodified for a given application, for example by use of either chemical,enzymatic or physical means to change the starting protein prior to orduring microparticle formation, or after formation of themicroparticles, matrices or after or during forming films or coatingsfrom the microparticles. Such modifications can produce microparticles,matrices, scaffolds, films or coatings with improved properties, such asaltered thermal stability, shear stability or resistance to proteases.Films or coatings made by such modifications would also have improvedfunctional properties including altered tensile and barrier properties,improved resistance to proteases and delayed biodegradation. Specificexamples of protein modifications are crosslinking with tannins, usingtrans glutaminase enzymes and heating.

The present invention is further described by the following examples.Such examples, however, are not to be construed as limiting in any wayeither the spirit or scope of the invention.

EXAMPLES Example 1 Formation of Kafirin Microparticles

A composition according to one embodiment of the invention was made byadmixing the following (all percentages are given on a weight basis): aplasticizer (10%), glacial acetic acid (66%) and kafirin (24%), and thekafirin was dissolved in the acid. Distilled water was then added withstirring, and microparticles were formed. The concentration of proteinwas 2%, plasticizer 40% in relation to the protein content and an acidconcentration of 5.4%.

The microparticles were prepared by weighing kafirin (1.8 g-88.63%protein) into a 125 ml Erlenmeyer flask. Plasticizer (0.66 g 1:1:1lactic acid, polyethylene glycol (400), glycerol-40% in relation toprotein) was mixed with glacial acetic acid (4.34 g) before adding tothe kafirin with gentle stirring. The temperature was slowly raised to30° C. to ensure full solvation. The protein solution was then leftovernight (16 h) at room temperature to relax the protein. After thisperiod distilled water was added slowly over a period of 5 min withstirring to a total weight of 80 g. On addition of the water themicroparticles formed, appearing as a stable, white colloidalsuspension. The particle size was from 1-10 microns, with the majorityof microparticles measuring 3-4 microns. There was no apparent increasein particle size with storage at 8° C. over a period of 6 months. Duringthis period, there was no microbial growth without any addedantimicrobial agents. When viewed by Scanning Electron Microscopy,microparticles appeared as spheres with extensively pitted surfaces(FIG. 2). Internal structure determined by Transmission ElectronMicroscopy revealed internal vacuoles of varying size with smooth walls.These results show that microparticles prepared by the process of thepresent invention have very large internal surface areas.

Example 2 Formation of Zein Microparticles Using Either Acetic Acid,Lactic Acid or Propionic Acid

A composition was made by admixing the following (all percentages aregiven on a weight basis): a plasticizer (9.5%), glacial acetic acid(62%) (or lactic acid or propionic acid) and commercial zein (ZPP Gold,Zein Protein Products, Marina, Calif.) (29.6%), and the zein wasdissolved in the acid. Distilled water was then added with stirring, andmicroparticles were formed. The concentration of protein was 10%,plasticizer 40% in relation to the protein content and an acidconcentration of 21.6%. To reduce the protein content further to 2%,21.6% acetic acid was added.

The microparticles were prepared by weighing zein (4 g-100% protein)into a 125 ml Erlenmeyer flask. Plasticizer (1.32 g 1:1:1 lactic acid,polyethylene glycol (400), glycerol-40% in relation to protein) wasmixed with glacial acetic acid (8.64 g) before adding to the zein withgentle stirring. The temperature was slowly raised to 30° C. to ensurefull solvation. The protein solution was then left overnight (16 h) atroom temperature to relax the protein. After this period distilled waterwas slowly over a period of 5 min with stirring to a total weight of 40g, 10% zein, 21.6% acetic acid. On addition of the water themicroparticles formed, appearing as a stable, white colloidalsuspension. To reduce the protein concentration further, 21.6% aceticacid was used for dilution.

Example 3 Formation of a Coating Made with Kafirin Microparticles andCrosslinked by Heating in Order to Protect the Coated Material e.g.Methionine, a Limiting Amino Acid, from the Conditions in the Rumen ofMulti-Gastric Animals

Microparticles of the invention were used to make a dried powder. Thispowder prepared as described above in the section, ‘powder formation’was mixed with a dry substance, in this case, the amino acid, methionineat a ratio of 1:1. The powder mixture was then mixed with glacial aceticacid (1:2) to form a paste with the aim of fusing together themicroparticles around the substance, e.g. methionine and forming acontinuous coating. The paste was then either dried directly on a flatsurface or first underwent a simple extrusion procedure without exposureto heat or excessive pressure. The coated material was then heat treatedin a force draught oven at 60-70° C. overnight. The microparticle coatedmethionine preparations were then subjected to dissolution and simulatedpepsin digestion assays to determine methionine release characteristics.

Incubation conditions for the dissolution test simulated the conditionsin the cow rumen with a pH of approximately 5.5 at 39° C. There was nota burst release of kafirin microparticle coated methionine. Insteadafter 8 h approximately 80% of the methionine was released from theglacial acetic acid fused microparticle coating compared with uncoatedmethionine, which was completely dissolved in less than 1 h under theseconditions. Release of methionine appeared to be by diffusion throughpores within the kafirin microparticle coating.

Thus, by coating the methionine or any other suitable compound with amicroparticle coating, a delayed release of such compound is effected.This allows the compound to bypass the rumen and be absorbed in theintestine of the animal as desired.

Example 4 Formation of a Free-Standing Film Made with KafirinMicroparticles

In another embodiment, microparticles of the invention were used to makea free-standing film. A microparticle suspension prepared as describedabove in Example 1 (4 g-2% protein, 5.4% acetic acid per film) wasweighed into plastic centrifuge tubes. The suspensions were centrifugedat 4000 g for 5 min. The supernatants were decanted off and replacedwith an equal weight of 21.6% acetic acid. The protein suspensions werethen left overnight at room temperature. Plasticizer (32 mg per film,mixed 1:1:1 lactic acid, polyethylene glycol (400), glycerol-40% inrelation to protein) was added to the protein suspension. The suspensionwas then cast into clean, dry flat plastic containers. The containerswere placed on a level surface in an oven (not forced draught) at 50° C.and dried for 4 hours. Films were gently peeled from the castingcontainers. Tensile and water barrier properties of the free standingfilms were then determined.

Free-standing films made from kafirin microparticles prepared by themethod described herein have significantly lower water vapourpermeability than films made with the same percentage kafirin usingconventional film casting techniques. Tensile properties of kafirinmicroparticle films were similar to those cast using conventional filmcasting techniques.

Kafirin microparticle films were much thinner (approximately 15 microns)(FIG. 4 a) than kafirin films (30 microns) of the same proteinconcentration cast using conventional film casting techniques (FIG. 4b). It is to be appreciated that the thickness of the free-standingmicroparticle films can be varied to obtain different properties.Similarly, the exact composition of the films can be varied, to impartdifferent properties to the resultant films.

In addition the films were clear, colourless and transparent.

Example 5 Formation of a Free-Standing Film Made with KafirinMicroparticles, Acid-Propionic Acid

Microparticles, of the invention were used to make a free-standing filmusing an alternative organic acid e.g. propionic acid. A microparticlesuspension prepared as described above in Example 1 (4 g-2% protein,5.4% propionic acid per film) was weighed into plastic centrifuge tubes.The suspensions were centrifuged at 4000 g for 5 min. The supernatantswere decanted off and replaced with an equal weight of 10.8% propionicacid. The protein suspensions were then left overnight at roomtemperature. Plasticizer (32 mg per film, mixed 1:1:1 lactic acid,polyethylene glycol (400), glycerol-40% in relation to protein) wasadded to the protein suspension. The suspension was then cast intoclean, dry flat plastic containers. The containers were placed on alevel surface in an oven (not forced draught) at 50° C. and dried for 4hours. Films were gently peeled from the casting containers (FIG. 5 e).Tensile, water barrier properties and protein digestibility of the freestanding films were then determined (Table 1).

Kafirin microparticle films cast from propionic acid were similar inthickness (approximately 15 microns) (FIG. 5 e) and appearance (clear,colourless and transparent) to kafirin microparticle films cast fromacetic acid (FIG. 5 c) and thinner than kafirin films (30 microns) ofthe same protein concentration cast using conventional film castingtechniques (FIG. 4 b).

TABLE 1 Functional properties of free-standing kafirin films 2% Kafirin2% Kafirin 2% Kafirin film microparticle microparticle cast from filmfilm (propionic glacial acetic Functional Properties (acetic acid) acid)acid Thickness (μm) 14.0 (3.0)* 14.0 (5.0) 30.0 (3.4) Water Vapour 0.22(0.05) 0.36 (0.11) 0.50 (0.11) Permeability (gmm/m²hkPa) Stress at break3.72 (4.87) 2.39 (4.13) 5.39 (2.07) (N/mm²) Elongation (%) 2.53 (3.37)1.15 (1.81) 2.99 (1.79) Protein digestion (%) 65.7 (2.5) 69.9 (6.7) 89.0(1.3) *FIGURES in parentheses indicate standard deviations

Example 6 Encapsulation of Dietary Antioxidants within KafirinMicroparticles for Monogastric Animals Including Humans

Microparticles of the invention were used to make a dried powder. Thispowder prepared as described above in the section, ‘powder formation’was mixed with a compound in solution, in this case, the polyphenolicantioxidants catechin or sorghum condensed tannins dissolved in 70%acetone, a solvent in which the microparticles are not soluble. Onevaporation of such solvent the compound remained encapsulated withinthe microparticles. The level of encapsulation was 20% polyphenol inrelation to protein. The examples used do not exclude the use of othersubstances or different solvents for those substances. The microparticleencapsulated antioxidants were then subjected to dissolution andsimulated digestion to determine the release profiles of theencapsulated antioxidants. Incubation conditions simulated those of thehuman digestive system, that is 2 h at 37° C. with pepsin at pH 2.0followed by a further 2 h incubation at the same temperature withtrypsin/chymotrypsin at pH 7.6.

After an initial small burst release, over a further period of fourhours, catechin and condensed tannin encapsulated kafirin microparticlesshowed virtually no protein digestion but released approximately 70% and50%, respectively, of their total antioxidant activity. Thisdemonstrates that encapsulation of polyphenolic antioxidants withinkafirin microparticles would allow controlled release of theencapsulated compound in the human stomach and gastrointestinal tract.It is to be appreciated that loadings of active compound could be variedin order to obtain different dosage levels. Similarly, the kafirinmicroparticles with the encapsulated compound may undergo furthertreatment, for example, by heat, in order to modify the release profileof the encapsulated compound.

Example 7 Extraction of Kafirin from Sorghum Brewers Spent Grain(Co-Product) and the Direct Preparation of Kafirin Microparticles

Sorghum brewers spent grain (moisture content 75%) was presoaked for 16h in a 0.5% aqueous solution of sodium metabisulphite at a solid tosolvent ratio of 1:5. The sodium metabisulphite was removed and thebrewers spent grain was extracted with glacial acetic acid at a solid tosolvent ratio of 1:5 for 48 h at ambient temperature. The extract(protein solution) was then separated from the solid material. The solidmaterial was washed with a further equal amount of glacial acetic acidand the two extracts combined. Microparticles were then made by the slowaddition of water to the glacial acetic acid extract (protein solution)with constant agitation. The ratio of extract (protein solution) towater added was 1:3 (the ratio of protein to acid to water isapproximately 2 parts protein, 25 parts acetic acid and 73 parts ofwater, depending on the original protein content of the brewers spentgrain). The resulting microparticles were then separated from the liquidand washed with water to remove any residual acid before drying. Thedried microparticles made by this method had a kafirin purity of 95% andcould then be used in any of the applications described in thisdocument.

Example 8 Direct Preparation of Kafirin Microparticle Films or Coatingsfrom an Extract of Sorghum Brewers Spent Grain

Microparticles made from sorghum brewers spent grain as described inexample 7 were used to make free-standing films and coatings. Instead ofwashing and drying the microparticles, the suspension of microparticlescan be taken directly and dried into a film or coating. In addition, ifdesired, the acid or protein concentration can be manipulated asdescribed in Example 4 in order to produce films and coatings ofdifferent thicknesses and with different functional properties (FIG. 6).Films made in this way were slightly thicker than those made fromisolated kafirin, being approximately 25 microns when 2% kafirin wasused and 15 microns when 1% kafirin was used. Lower proteinconcentrations resulted in coatings which could not be separated fromthe casting surface.

Films made from this and other co-products were usually slightly cloudybut still complete. The cloudiness is probably due to the impuritiesco-extracted with the kafirin. Making microparticles from pure kafirinand then using these microparticles to make films we get clear,transparent films which are complete.

Example 9 Use of a Kafirin Microparticle Coating Formulation to Extendthe Shelf Life of Minimally Processed Avocados

Microparticles made from sorghum brewers spent grain as described inExample 7 were used to make a coating formulation which was applied tocut halves of eat-ripe avocados to extend the fruits' shelf life. Class1 export avocados (Persea americana Mill) variety ‘Hass’ were used inthe experiment. The avocados were stored at 4° C. 5 days prior to thestart of the experiment. The avocados were then ripened at 22° C. untileat-ripe. They were peeled and halved by hand before dipping in thecoating formulation solution. The coating formulation solution consistedof 2% kafirin protein in 2% (v/v) acetic acid with 1:1:1 plasticiser ofpropylene glycol, lactic acid and glycerol (40% w/w with respect toprotein). Uncoated avocado halves were used as controls. After thecoating had dried, the fruit halves were packaged in plastic tubs, whichwere closed with plastic lids, and stored at 4° C. for 5 days. After theperiod of 5 days, the microparticle-coated avocados showed no evidenceof browning, whereas the control avocados showed extensive browning(FIG. 7).

While the invention has been described in detail with respect tospecific embodiments thereof, it will be appreciated by those skilled inthe art that various alterations, modifications and other changes may bemade to the invention without departing from the spirit and scopethereof.

1. A process for making protein microparticles, the process includingthe steps of: (a) dissolving cereal prolamin protein in an organic acidwith agitation and in the absence of an alcohol; and (b) diluting thesolution containing the protein with an aqueous solution, therebyforming protein microparticles having vacuoles.
 2. A process accordingto claim 1, wherein the cereal prolamin protein is selected from thegroup consisting of gluten, hordein, zein, kafirin, and combinationsthereof.
 3. A process according to claim 1, wherein the organic acid isselected from the group consisting of propionic acid, lactic acid,acetic acid, and combinations thereof.
 4. A process according to claim1, which is carried out in the absence of ethanol.
 5. A processaccording to claim 1, wherein in step (a) the protein solution issaturated or highly concentrated.
 6. A process according to claim 1,wherein the agitation is provided by stirring, shaking, homogenization,or bubbling gas through the solution.
 7. A process according to claim 1,wherein the protein is crosslinked with tannins or enzymes and/or heattreated before or after step (a).
 8. A process according to claim 1,which further includes a step of removing the organic acid from thediluted solution.
 9. A process according to claim 1, which furtherincludes a drying step.
 10. A process according to claim 1, whichfurther includes a step of forming a powder, film, coating, matrix, orscaffold from the diluted solution.
 11. A process according to claim 1,wherein the solution in step (a) is allowed to stand for 8 to 24 hours.12. A process according to claim 1, wherein in step (b) the solutioncontaining the protein is diluted with water to a final concentration ofabout 2 parts by weight of the protein to about 2 to about 30 parts byweight of the organic acid to about 96 to about 68 parts by weight ofwater.
 13. A process according to claim 1, wherein an additive, selectedfrom the group consisting of a plasticizer, colouring agent, flavouringagent, preservative, trace mineral, nutrient, enzyme, hormone,nutraceutical, probiotic, prebiotic, pharmaceutical, and combinationsthereof, is added to the solution of step (a) or (b) and is incorporatedinto or encapsulated by the protein microparticles.
 14. A processaccording to claim 13, wherein the additive is a plasticizer selectedfrom the group consisting of glycerol, polyethylene glycol,polypropylene glycol, lactic acid, dibutyl tartrate, monoglycerides,palmitic acid, stearic acid, triethylene glycerol, sorbitol, andcombinations thereof.
 15. A process according to claim 14, wherein theplasticizer is present in an amount of from about 0.2 to about 0.6 partsby weight to about 1 part by weight of the protein.
 16. A proteinmicroparticle prepared according to the process of claim
 1. 17. Aprotein microparticle according to claim 16, which has a vacuole.
 18. Apowder comprising protein microparticles of claim
 17. 19. An aqueoussuspension comprising protein microparticles of claim
 17. 20. A filmformed from protein microparticles of claim
 17. 21. A film according toclaim 20, which is complete.
 22. A coating formed from proteinmicroparticles of claim
 17. 23. A matrix formed from proteinmicroparticles of claim
 17. 24. A scaffold formed from proteinmicroparticles of claim
 17. 25. A substrate including the coating ofclaim 22.